Mycopathologia (2007) 164:241–248
DOI 10.1007/s11046-007-9057-0
Inhibition of ochratoxin A production and growth
of Aspergillus species by phenolic antioxidant compounds
Jeffrey D. Palumbo Æ Teresa L. O’Keeffe Æ
Noreen E. Mahoney
Received: 15 December 2006 / Accepted: 29 August 2007 / Published online: 12 September 2007

Springer Science+Business Media B.V. 2007
Abstract The phenolic antioxidants, gallic acid,
vanillic acid, protocatechuic acid, 4-hydroxybenzoic
acid, catechin, caffeic acid, and chlorogenic acid
were studied for their effects on ochratoxin A (OTA)
production and fungal growth of ochratoxigenic
Aspergilli. Of the 12 strains tested, which included
A. alliaceus, A. lanosus, A. ochraceus, A. albertensis,
A. melleus, A. sulphureus, A. carbonarius, A. elegans,
and A. sclerotiorum, the greatest inhibition of OTA
production was seen in A. sulphureus, A. elegans, and
A. lanosus. Vanillic acid and 4-hydroxybenzoic acid
were the most inhibitory to both OTA production and
growth of most of the strains tested. However,
A. ochraceus was not inhibited by either compound,
and A. carbonarius was not inhibited by vanillic acid.
The effect of each compound on OTA production and
growth differed among strains and generally was
variable, suggesting that species-specific OTA pro-
duction and response to phenolic compounds may be
influenced by different ecological and developmental
factors. In addition, inhibition of OTA production by
antioxidant compounds may be useful in determining
biosynthetic and regulatory genes involved in both
OTA production and stress response in ochratoxi-
genic Aspergilli.
J. D. Palumbo (&)
T. L. O’Keeffe
N. E. Mahoney
Plant Mycotoxin Research Unit, U.S. Department
of Agriculture, Agricultural Research Service,
800 Buchanan St., Albany, CA 94710, USA
e-mail: palumbo@pw.usda.gov
Keywords Fungal inhibition

Mycotoxin inhibition
Ochratoxigenic Aspergillus

Ochratoxin A
Phenolic antioxidants
Introduction
Ochratoxin A (OTA) is a polyketide mycotoxin com-
posed of a chlorinated dihydro-methyl-isocoumarin
moiety linked via an amide bond to phenylalanine [1].
OTA is a secondary metabolite produced by several
Aspergillus species, including but not limited to
A. ochraceus, A. alliaceus, A. auricomus, A. sulphureus,
A. carbonarius, A. niger, and A. glaucus, as well as
Penicillium verrucosum and P. nordicum [2–8]. OTA
has been shown to be nephrotoxic, carcinogenic,
teratogenic, and immunosuppressive [3, 9–11]. In
addition, OTA has been shown to induce oxidative
DNA damage in mammalian cells and in rat liver and
kidney in vivo, indicating its role in cytotoxicity and
tumorigenicity [12, 13]. Food products associated with
OTA contamination include cereal grains, beer, wine,
grapes, coffee, and dried fruit and nuts [2, 3, 14–20]. As
a result of the wide variety of sources of this toxin,
regulatory limits for OTA content in commodities have
been set in the European Union [21].
Fungal production of OTA has been shown to be
affected by environmental and nutritional factors,
such as pH, temperature, water activity, and carbon
and nitrogen sources [22–25]. Food additives includ-
ing sodium propionate, methyl paraben, sodium
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bisulfite, and potassium sorbate were shown to inhibit
OTA production in A. sulphureus [26], but there have
been no other studies involving the effects of chemical
natural products on OTA biosynthesis. In addition,
because the biochemical and genetic components of
the OTA biosynthetic pathway is not fully understood,
and because of the species diversity of OTA produc-
ers, molecular studies regarding the regulation of
OTA production have been limited [24, 25].
Recently, several antioxidant compounds have been
shown to inhibit aflatoxin biosynthesis in A. flavus and
A. parasiticus [27–30], and several genetic mecha-
nisms involving oxidative stress-responsive genes have
been identified [31, 32]. We hypothesized that OTA
production in ochratoxigenic Aspergillus species would
be affected similarly by phenolic antioxidants. Using a
defined medium, the effects of these compounds on
OTA production and fungal growth were investigated,
as a first step in determining whether ochratoxigenesis
is dependent on similar biochemical and genetic
mechanisms as aflatoxigenesis.
Materials and methods
Ochratoxigenic fungal strains
A. alliaceus NRRL315 (isolated from blister beetle),
A. albertensis NRRL20602 (isolated from human
ear), A. melleus NRRL3520 (of unknown origin),
A. sulphureus NRRL4077 (isolated from soil), and
A. carbonarius NRRL369 (isolated from paper) were
obtained from the USDA Agricultural Research
Service (NRRL) Culture Collection (Peoria, IL).
A. ochraceus ATCC22947 (isolated from sorghum)
was obtained from the American Type Culture
Collection (Manassas, VA). Other fungal strains used
in this study were isolated previously from different
sources (P. Bayman and J. Baker, unpublished).
A. alliaceus O-106 was isolated from a Joshua tree
(Yucca brevifolia) flower. A. lanosus strains 114 and
171 and A. sclerotiorum O-196 were isolated from
pistachio orchard soil. A. ochraceus R46 was isolated
from coffee borer beetle. A. elegans J-93 was isolated
from green coffee. Strains were grown on potato
dextrose agar (PDA) (Difco, Becton Dickinson,
Franklin Lakes, NJ) at 28C until conidia developed,
and conidial stock suspensions were made in 30%
glycerol, 0.05% Tween 80 for storage at –70C.
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Mycopathologia (2007) 164:241–248
Media and growth conditions
Experiments were performed using Aspergillus mini-
mal medium (AMM) [33] containing 100 mM glucose
and 70 mM NH4Cl as carbon and nitrogen sources,
respectively. For A. ochraceus strains, AMM was
supplemented with 1% (wt vol–1) yeast extract. Gallic
acid (3,4,5-trihydroxybenzoic acid), vanillic acid
(4-hydroxy-3-methoxybenzoic acid), protocatechuic
acid (3,4-dihydroxybenzoic acid), 4-hydroxybenzoic
acid, (+) catechin, caffeic acid (3,4-dihydroxycinnamic
acid), and chlorogenic acid (all from Sigma-Aldrich,
St. Louis, MO) were added individually to AMM and
the media were sterilized by filtration using 0.2 lm
cellulose nitrate filters (Nalge Nunc International,
Rochester, NY). Fungal cultures were incubated at
28C without shaking.
Effect of antioxidant concentration on OTA
production by A. alliaceus
AMM was prepared with 0, 1.25, 2.5, 5, 10, or 20 mM
of each antioxidant compound, and 1-ml aliquots were
dispensed in triplicate into 24-well culture plates.
Wells were inoculated with 10 ll of A. alliaceus strain
O-106 conidial suspension (*105 conidia ml–1) and
incubated at 28C for 5 days. OTA in 100 ll of each
culture was partitioned into 100 ll of chloroform,
which was dried under a stream of nitrogen gas and
redissolved in 10 ll of methanol. Samples (1 ll each)
were spotted onto 0.5-mm-thick silica gel thin-layer
chromatography (TLC) plates and separated using a
mobile phase of toluene:ethyl acetate:formic acid
(5:4:1). OTA spots were visualized under ultraviolet
light in comparison to authentic OTA (Sigma-
Aldrich), and spot intensities were determined using
ImageJ image analysis software [34]. Mean OTA
content in triplicate cultures of each treatment was
calculated relative to untreated triplicate cultures.
Quantification of OTA production and growth
of Aspergillus species
Each antioxidant compound was added to AMM at a
concentration of 10 mM. Ten ml of each medium
were added to 60 mm diameter Petri dishes and
inoculated with 10 ll of conidial suspensions
(*105–106 conidia ml–1) of each Aspergillus strain
Mycopathologia (2007) 164:241–248 243

described above. Plates were sealed with parafilm and
incubated at 28C for 6 days. Following incubation,
3 ml of culture liquid from each plate were filtered
using 0.22 lm Millex-GP polyethersulfone syringe
filters (Millipore, Bedford, MA). OTA in the culture
filtrates was partitioned into 3 ml of chloroform. The
chloroform phase was collected, evaporated to dry-
ness under a stream of nitrogen gas and redissolved in
1 ml of HPLC-grade methanol. Samples were ana-
lyzed by injection of 20 ll into an Agilent model 1100
high-performance liquid chromatograph with model
1321A fluorescence detector (Agilent Technologies,
Inc., Santa Clara, CA). Separations were run on a
Vydac 218TP54, 4.6 · 250 mm C18 column (Grace
Vydac, Hesperia, CA) with methanol:water:phospho-
ric acid (70:30:0.1) as the mobile phase at a flow rate
of 1 ml min–1. OTA was detected using excitation and
emission wavelengths of 333 nm and 418 nm, respec-
tively. OTA amounts per sample (3 ml culture filtrate)
were calculated from peak areas in comparison to a
standard curve constructed using 5–300 ng of authen-
tic OTA (Sigma-Aldrich) per injection. Total OTA
per plate was calculated as OTA per sample · 3.33.
From the same cultures, fungal mycelium was recov-
ered by filtration onto Whatman no. 4 paper filters and
air dried overnight for dry weight determination.
Specific OTA production was calculated as ng OTA/
mg fungal dry weight.
Statistical analyses
All treatments for quantification were performed in
triplicate. Data were analyzed using GraphPad InStat
version 3.06 (GraphPad Software, San Diego, CA).
One-way ANOVA with Dunnett’s posttest was
performed to compare specific OTA production and
fungal dry weights in antioxidant-treated samples to
untreated samples.
Results and discussion
Previous efforts in our laboratory to understand OTA
production focused on California isolates of
A. alliaceus. Initial experiments showed that OTA
production by A. alliaceus strain O-106 was inhibited
in response to increasing concentrations of phenolic
antioxidants. As shown in Table 1, most of the
compounds inhibited the total OTA content relative
to control cultures at concentrations of 10 mM or
higher, with the exception of catechin. We therefore
tested these compounds for OTA inhibition in other
Aspergillus species at 10 mM.
Table 2 shows the effects of the antioxidant
compounds we tested on OTA production by nine
OTA-producing Aspergillus species. In the 12 strains
tested, OTA content of untreated cultures was highly
variable among triplicate samples, leading to large
standard deviations, which in some cases precluded
meaningful statistical analyses of antioxidant treat-
ments relative to controls. This variability has been
attributed by other researchers investigating OTA
production to medium effects [23], and our own
observations with OTA suggest that minimizing
variability requires more thorough investigation of
potential causes. These challenges are also inherent in

Table 1 Antioxidant effects on OTA production in A. alliaceus O-106a

Treatment Antioxidant concentration (mM)
0 1.25 2.5 5 10 20

Gallic acid 100 (26) 150 (11) 134 (4) 128 (2) 68 (26) 74 (18)
b
Vanillic acid 100 (16) 154 (2) 134 (5) 59 (6) n.d. n.d.
Protocatechuic acid 100 (14) 117 (4) 128 (5) 115 (2) 65 (5) 24 (10)
4-Hydroxybenzoic acid 100 (6) 100 (6) 92 (7) 71 (30) 30 (5) 16 (2)
Catechin 100 (12) 130 (25) 134 (7) 124 (46) 143 (4) 97 (26)
Caffeic acid 100 (15) 133 (3) 118 (8) 101 (18) 52 (17) 28 (5)
Chlorogenic acid 100 (3) 83 (4) 79 (3) 70 (10) 25 (6) 16 (6)
a
Mean OTA production per fungal culture, expressed as percent relative to untreated cultures. Numbers in parentheses represent
standard deviation of three replicate treatments
b
n.d.—not detected

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244 Mycopathologia (2007) 164:241–248

Table 2 Effect of phenolic antioxidants on OTA production in Aspergillus sppa

Treatment A. alliaceus A. alliaceus A. lanosus A. lanosus A. ochraceus A. ochraceus
NRRL315 O-106 114 171 ATCC22947 R46

Control 118 (73.3) 67.3 (35.0) 64.7 (40.4) 78.8 (38.0) 107 (35.1) 642 (143)
b b,c b
Gallic acid 44.7 (27.1) 14.3 (4.7) n.d. 0.7 (0.5) 199 (222) 378 (26.1)
Vanillic acid 22.9 (39.6)b 1.6 (0.6)b n.d.b n.d.b 143 (204) 732 (978)
Protocatechuic acid 79.1 (51.2) 25.8 (10.5)b 0.3 (0.2)b 1.4 (0.8)b 418 (363) 125 (130)
4-Hydroxybenzoic acid n.d.b 5.1 (2.7)b n.d.b n.d.b 345 (397) 798 (556)
Catechin 71.0 (16.3) 53.4 (2.6) 24.6 (7.3)b 15.8 (5.2)b 19.7 (16.7) 14.5 (8.1)
b b b b
Caffeic acid 10.0 (4.8) 13.1 (10.4) n.d. 1.6 (0.2) 344 (292) 293 (54.2)
Chlorogenic acid 29.5 (5.7)b 6.5 (1.3)b 0.1 (0.2)b 0.6 (0.3)b 95.7 (113) 110 (98.2)
Treatment A. albertensis A. melleus A. sulphureus A. carbonarius A. elegans A. sclerotiorum
NRRL20602 NRRL3520 NRRL4077 NRRL369 J-93 O-196
Control 184 (91.2) 239 (84.4) 150 (36.6) 19.3 (16.5) 151 (43.7) 641 (313)
Gallic acid 60.5 (32.1)b 46.1 (22.7) n.d.b 3.5 (4.2) n.d.b 1.8 (0.7)b
Vanillic acid n.d.b 13.5 (7.6) n.d.b 25.3 (30.5) n.d.b n.d.b
b b b
Protocatechuic acid 33.1 (19.7) 35.8 (0.7) 2.4 (2.6) 16.1 (13.8) 0.2 (0.0) 26.7 (9.6)b
b b b
4-Hydroxybenzoic acid 6.2 (2.3) 7.5 (5.8) n.d. 1.5 (1.1) 1.7 (0.4) 17.2 (29.8)b
Catechin 18.6 (6.6)b 1580 (296)d 0.4 (0.2)b 29.6 (12.9) 62.0 (105) 5.5 (3.3)b
Caffeic acid 31.8 (10.6)b 18.2 (6.3) n.d.b 9.2 (14.3) n.d.b 6.4 (1.5)b
Chlorogenic acid 26.4 (7.0)b 563 (186)d 6.3 (9.6)b 6.3 (7.4) n.d.b 136 (50.6)b
a
Mean OTA content, expressed as ng OTA/mg fungal dry weight. Numbers in parentheses represent standard deviation of three
replicate samples per treatment
b
Significant reduction of OTA content relative to control, P \ 0.05
c
n.d.—not detected
d
Significant increase in OTA content relative to control, P \ 0.05

analyses of aflatoxin production by different A. flavus
strains (R. Molyneux and N. Mahoney, personal
communication). Nevertheless, the data indicate
consistent trends of OTA inhibition among treat-
ments, which are illustrated in the ranges of inhibition
among triplicate samples.
OTA production by A. alliaceus strains NRRL315
and O-106 was inhibited strongly by 4-hydroxyben-
zoic acid (94–100%), caffeic acid (63–96%), and
chlorogenic acid (70–90%). Vanillic acid (42–100%),
protocatechuic acid (40–74%), and gallic acid
(36–86%), also showed OTA inhibition in both
A. alliaceus strains. Catechin (17–56%) was gener-
ally less effective at inhibiting OTA production in
these strains. Similarly, A. albertensis exhibited
strong OTA inhibition, most effectively by vanillic
acid (100%) and 4-hydroxybenzoic acid (96–98%),
followed by catechin (87–94%), chlorogenic acid
(83–90%), caffeic acid (79–89%), protocatechuic
acid (72–93%), and gallic acid (57–87%).
OTA production by A. lanosus strains 114 and 171
was inhibited strongly by all the compounds tested.
Other than catechin, which inhibited toxin production
by 52–87%, all the other compounds inhibited 97–
100% of the toxin production by these strains. These
effects were also shown in the A. sulphureus and
A. sclerotiorum strains tested, showing 88–100% and
74–100% OTA inhibition, respectively. In A. elegans,
OTA production was inhibited by all the compounds
tested by 99–100%, with the exception of a single
catechin treatment that showed OTA production at
the level of the control treatments.
A. melleus showed more variable inhibition by
antioxidant compounds. Vanillic acid (91–97%),
4-hydroxybenzoic acid (94–99%), and caffeic acid
(90–95%) tended to inhibit OTA production most
strongly. Intermediate levels of inhibition were seen
for protocatechuic acid (85%) and gallic acid (70–
88%). In contrast, A. melleus cultures grown on
AMM containing catechin produced 5- to 8-fold more

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Mycopathologia (2007) 164:241–248
OTA than control cultures, and on AMM containing
chlorogenic acid produced 1.6- to 3-fold more OTA
than control cultures.
The effects of the antioxidant compounds on
A. carbonarius were also highly variable. Although
the results were not statistically significant, gallic
acid, 4-hydroxybenzoic acid, and chlorogenic acid
tended to inhibit OTA production. In contrast,
vanillic acid, caffeic acid, protocatechuic acid, and
catechin produced widely divergent effects among
replicate samples, ranging from 97% OTA inhibition
to 3-fold higher OTA levels relative to control
treatment in individual samples. It should be noted
that in AMM containing glucose and ammonium,
control cultures of A. carbonarius produced very low
levels of OTA relative to the other strains tested in
this study, which may contribute to the high
variability in its response to antioxidant treatments.
In contrast to the other strains tested in this study,
A. ochraceus strains ATCC22947 and R46 grew
slowly and did not produce OTA on AMM containing
glucose and ammonium (data not shown). Therefore,
yeast extract was added to the media for growth of
these strains, to promote OTA production and more
vigorous growth. However, this tended to increase the
variability in OTA production among replicate sam-
ples. Although treatments were not significantly
different, trends among treatments were apparent, as
shown in Table 2. A. ochraceus strain R46 produced
higher levels of OTA than A. ochraceus strain
ATCC22947. Also, antioxidant compounds generally
were more inhibitory to OTA production in A. ochrac-
eus strain R46 than in A. ochraceus strain ATCC22947.
Catechin, chlorogenic acid, and protocatechuic acid
tended to strongly inhibit OTA production in strain
R46. Lower amounts of OTA inhibition were seen with
caffeic acid and gallic acid. In contrast, the response of
strain R46 to vanillic acid and 4-hydroxybenzoic acid
was inconsistent among replicates, with a range of 82%
inhibition to 3-fold increase and 53% inhibition to
2-fold increase, respectively, in OTA production.
A. ochraceus strain ATCC22947 generally showed
no consistent OTA inhibition in response to any of the
antioxidants tested, with the exception of catechin,
which inhibited OTA production by 70–99%. The
differences in OTA levels produced by control cultures
and response to antioxidants between A. ochraceus
strains may be due in part to laboratory domestication
[7], i.e., strain ATCC22947 was isolated in 1972 [35]
245
and has been subcultured many times, while strain R46
was isolated in 2002 and subcultured only a few times.
Also, since OTA production generally is higher on
complex media, growth of A. ochraceus on AMM
supplemented with yeast extract may have made the
inhibitory effects of the compounds difficult to discern.
In addition to the effects on OTA production,
antioxidant compounds also affected fungal growth
(Table 3). A. alliaceus strain NRRL315 was inhibited
most strongly by vanillic acid and 4-hydroxybenzoic
acid, and significant effects on growth were seen for
all other compounds except catechin. In contrast,
growth of A. alliaceus strain O-106 was inhibited only
by vanillic acid. Interestingly, in strain O-106,
4-hydroxybenzoic acid, chlorogenic acid, caffeic acid,
and gallic acid inhibited OTA production (Table 2)
with no inhibition of fungal growth (Table 3). As with
A. ochraceus strains, strain differences in this case
may be due to the laboratory domestication of
A. alliaceus strain NRRL315 and the relative wildness
of strain O-106.
Growth was inhibited in both A. lanosus strains
most strongly by vanillic acid and 4-hydroxybenzoic
acid. Like A. alliaceus strain O-106, A. lanosus growth
was less affected by the other antioxidants tested
(although the reduction was statistically significant), at
the same time OTA production was strongly inhibited.
Similarly, gallic acid and chlorogenic acid did not
affect growth of A. carbonarius but tended to inhibit
OTA production. In A. elegans, treatment with gallic
acid, 4-hydroxybenzoic acid, and caffeic acid inhib-
ited OTA production without affecting growth, and
treatment with protocatechuic acid and chlorogenic
acid resulted in significantly higher biomass than
controls and strong inhibition of OTA production.
Growth of A. sulphureus was significantly reduced
by all compounds tested except catechin, which
increased fungal biomass significantly while inhibit-
ing OTA production. Similarly, A. sclerotiorum
growth was significantly inhibited by gallic acid,
vanillic acid, 4-hydroxybenzoic acid, and chlorogenic
acid, which also inhibited OTA production. OTA
production, but not growth, was inhibited by cate-
chin, while caffeic acid affected OTA production
more strongly than growth, although growth inhibi-
tion was significant. A. albertensis growth was
reduced only by vanillic acid and 4-hydroxybenzoic
acid, which were also the strongest inhibitors of OTA
production. A. melleus growth was affected by gallic
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246 Mycopathologia (2007) 164:241–248

Table 3 Effect of phenolic antioxidants on growth of Aspergillus sppa

Treatment A. alliaceus A. alliaceus A. lanosus A. lanosus A. ochraceus A. ochraceus
NRRL315 O-106 114 171 ATCC22947 R46

Control 60.5 (6.9) 60.3 (4.4) 61.4 (5.4) 62.0 (4.7) 195.2 (6.3) 199.5 (2.1)
b b
Gallic acid 32.7 (8.9) 63.6 (1.3) 57.7 (6.6) 49.1 (5.6) 202.7 (8.7) 210.6 (5.4)
Vanillic acid 1.6 (0.2)b 17.8 (2.7)b 7.8 (2.5)b 6.4 (0.2)b 134.4 (41.1)b 139.4 (39.2)b
Protocatechuic acid 45.6 (4.8)b 61.5 (3.3) 47.5 (1.8)b 49.8 (8.0)b 191.1 (20.6) 189.7 (24.1)
b b
4-Hydroxybenzoic acid 2.6 (1.1) 54.5 (2.1) 8.0 (0.6) 7.9 (0.4)b 184.0 (15.8) 206.3 (16.0)
Catechin 63.5 (1.7) 73.6 (5.2)c 67.0 (3.4) 66.9 (3.4) 220.4 (2.6) 197.0 (35.3)
b b
Caffeic acid 29.1 (9.5) 59.8 (1.7) 56.9 (1.2) 49.1 (3.3) 195.7 (14.5) 221.2 (1.7)
Chlorogenic acid 21.6 (5.9)b 68.2 (3.0)c 47.7 (4.4)b 43.1 (7.4)b 218.3 (11.9) 240.6 (11.4)
Treatment A. albertensis A. melleus A. sulphureus A. carbonarius A. elegans A. sclerotiorum
NRRL20602 NRRL3520 NRRL4077 NRRL369 J-93 O-196
Control 65.4 (4.1) 35.3 (2.6) 25.2 (3.3) 109 (1.3) 23.3 (0.4) 40.0 (3.3)
Gallic acid 72.0 (9.7) 26.2 (7.1)b 1.1 (0.5)b 109 (10.6) 28.8 (3.4) 6.2 (0.6)b
Vanillic acid 4.5 (1.2)b 20.4 (2.0)b 1.6 (0.5)b 90.2 (28.4) 12.8 (0.5)b 4.9 (0.4)b
b c
Protocatechuic acid 70.7 (7.1) 30.1 (1.7) 2.1 (0.9) 112 (4.8) 40.4 (3.0) 36.0 (3.1)
4-Hydroxybenzoic acid 25.5 (5.5)b 30.7 (2.1) 3.1 (0.8)b 67.8 (9.3)b 31.3 (2.6) 5.5 (0.8)b
Catechin 74.9 (5.3) 30.9 (3.3) 34.3 (1.3)c 112 (13.4) 8.6 (5.3)b 37.7 (5.0)
Caffeic acid 65.1 (2.9) 35.9 (1.0) 11.6 (3.2)b 100 (11.7) 30.6 (4.0) 31.1 (2.4)b
Chlorogenic acid 64.0 (6.9) 26.7 (2.1)b 5.1 (0.8)b 120 (15.0) 41.6 (7.1)c 9.0 (2.2)b
a
Mean fungal growth, expressed as mg dry weight. Numbers in parentheses represent standard deviation of three replicate samples
per treatment
b
Significant reduction of fungal dry weight relative to control, P \ 0.05
c
Significant increase in fungal dry weight relative to control, P \ 0.05

acid, vanillic acid, and chlorogenic acid, but not by
OTA-inhibiting 4-hydroxybenzoic acid.
Growth of A. ochraceus strains in AMM supple-
mented with yeast extract was significantly inhibited
by vanillic acid, but not by the other compounds
tested. A. ochraceus strain ATCC22947 showed
similar results in AMM without yeast extract, while
growth of strain R46 was reduced by vanillic acid,
protocatechuic acid, 4-hydroxybenzoic acid, and cat-
echin in AMM without yeast extract (data not shown).
Differences in growth response to antioxidant com-
pounds between A. ochraceus strains, like differences
in OTA production, may be in part due to differences
in laboratory domestication, as discussed above.
Conclusions
Data presented in this study indicate that antioxidants
generally suppress OTA production in several impor-
tant ochratoxigenic Aspergillus species. All of the
antioxidant compounds tested in this study were
inhibitory to one or more Aspergillus strain tested,
and vanillic acid, and 4-hydroxybenzoic acid were
the most effective inhibitors of OTA production and
growth of many of these strains. These data suggest
that the mechanisms of phenolic antioxidant activity
may be directly or indirectly related to primary
metabolism, as evidenced by effects on fungal
growth, or involved in secondary metabolism, or a
combination of the two. Further experimentation will
be necessary to determine whether the effects on
production of secondary metabolites, such as OTA
are due to effects on the oxidative stress response of
the fungus. Also, OTA inhibition by these com-
pounds may be useful in determining differences in
gene expression in response to the compounds, and to
identify biosynthetic and regulatory genes necessary
for OTA production. Additional studies to determine
dose-response effects of inhibitory compounds on
OTA production in a broad range of ochratoxigenic
Aspergillus species will be useful in indicating

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effective inhibitory concentrations for potential use in
preventative intervention strategies.
Phenolic antioxidant compounds have been studied
in coffee [36], grapes and wine [37], barley [38, 39],
and tree nuts including walnut [28, 40], almond [41,
42], and pistachio [43, 44]. These antioxidant sources
are also common habitats of ochratoxigenic Aspergillus
and Penicillium species, leading to potential OTA
contamination of these commodities. This apparent
contradiction indicates that the role of antioxidants and
OTA production in the ecology of ochratoxigenic fungi
is complex. Our results show that individual antioxi-
dants have variable effects on both OTA production
and growth of individual ochratoxigenic Aspergillus
strains and that these effects may or may not be
independent of each other. As a result, further inves-
tigations regarding bioavailability and fungal response
to phenolic and polyphenolic antioxidants under eco-
logically relevant conditions is warranted. Information
regarding genetic and physiological responses to
antioxidant compounds could lead to targeted inter-
vention strategies for the reduction of economic losses
by OTA contamination.
Acknowledgments We thank J. H. Kim for helpful
discussions, and L. Gorski for careful review of the
manuscript. This work was supported by U.S. Department of
Agriculture, Agricultural research Service CRIS project no.
5325-42000-035-00D, and Foreign Agriculture Service
Scientific Cooperation Research Program grant no. 5325-
42000-035-02.
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